The purpose of this proposal is to study the biochemistry and enzymology of thyroid hormone metabolism in target tissues. It is well established that extrathyroidal conversion of thyroxine (T4) to 3,5,3 feet-tiiodothyronine (T3) is the major source of circulating bioactive hormone thus serving as an initiating step in the mechanism of action of thyroid hormone in man and animals. Recent studies have shown that target tissues differ widely in their abilities to produce T3 from T4 and in their abilities to utilize the locally produced bioactive hormone. These tissue specific differences in hormone metabolism result, in part, from the presence of two isozymes of iodothyronine 5 feet-deiodinase, the enzyme catalyzing T4 to T3 conversion. These differ in reaction kinetics, substrate specificities and the influences of disease states on catalysis. The identities and inter-relationships between these two isozymes are, however, poorly understood. Iodothyronine 5 feet-deiodinase will be purified from catalytically active detergent extracts of renal membranes by ion-exchange and affinity chromat ography and membrane and phospholipid requirements, hydrodynamic properties and the immunological identity determined. Selective chemical modification of active center sulfhydryls and photo-affinity labeling using derivatized substrates and inhibitors will allow purification of enzyme polypeptides based on specific labeling rather than catalytic properties. Fragmentation of the specifically labeled enzyme will provide a partial library of enzyme-related peptides which will aid in the development of site-specific anti-enzyme antibodies. Anti-enzyme antibodies will be produced by hybridoma methodology from purified, functionally competent enzyme preparations and specifically labeled enzyme protein and peptide fragments. Similarities between the ligand binding site of anti-substrate antibodies and the substrate binding site of the 5 feet-deiodinase will be used to produce anti-rT3 IgG antiidiotypic monoclonal antibodies which recognize the renal iodothyronine 5 feet-deiodinase. The availability of site-specific anti-enzyme antibodies will allow definitive characterization of the anatomical and ultrastructural distribution of the enzyme and will clarify the inter-relationships between the tissue-specific isozymes of 5 feet-deiodinase.